Natural Killer (NK) cells have been implicated in defense against cancer, viral infections and regulation of hemopoiesis. The longer term objective of this proposal is to investigate the lineage and sequential differentiation of NK cells from pluripotent lympho-hemopoietic stem cells and to understand the function of NK-associated molecules. To accomplish this we will purify IL-2 unresponsive transplantable NK progenitor (NKp) cells from mouse bone marrow, by multiparameter cell sorting. Populations enriched for Nkp activity (e.g. Ly6+, Lineage-, CD43+, Fall 3+) will be tested for pluripotent stem cells, and transplantable T and B cell progenitors. The T and B progenitor assays will be performed by adoptive transfer of cells into irradiated SCID mice. Marrow populations co-enriched for NKp and other progenitor cells will be subdivided phenotypically to separate committed NKp from pluripotent stem cells and other lymphoid and myeloid progenitors. Flow cytometry based enrichment and separation will also be performed with cells obtained from adult and fetal thymus, to determine if NK cells are derived from a common lymphoid stem cell or bipotential NK/T progenitor cells. Particular emphasis will be placed markers such as Joro-75 believed to recognize committed T progenitors. If a candidate NK/T progenitor population if found, we will to test if T cells and NK cells can be derived from progeny of single sorted cells. IL-2 responsive committed 3A4+ NK-1.1-NK precursor cells will also be purified by flow cytometry. To determine whether these NK precursor cells respond to IL-2 directly or via accessory cells, the purified cells will be cultured with IL-2 and expression of IL-2Rbeta on 3A4+, NK-1.1- cells will also be determined. The sequential development of NK cells from pluripotent stem cells will be tested in long term bone marrow cultures. We will test the hypothesis that pluripotent stem cells differentiate into NK progenitor cells and then to IL-2 responsive NK precursors under the influence of distinct stromal microenvironments. Ultimately, an attempt will be made to recreate the entire sequence of NK cell differentiation by sorting limited numbers of purified progenitor cells on defined stromal cells. To understand the function of the 3A4 molecule expressed on immature IL-2 responsive NK precursor we will clone and sequence 3A4 cDNA. We discovered that 2B4 an NK-associated molecule is expressed on all cells that mediate non-MHC-restricted (NK-like) cytotoxicity after culture in IL-2 and we have cloned 2B4 cytotoxicity by anti-sense and transfection techniques. An understanding of the early steps in NK cell differentiation and the function of molecules expressed on immature and mature NK cells may allow the modulation of NK function for cancer immunotherapy.